Antibody combinations and use of same for treating cancer

ABSTRACT

A method of identifying a combination of antibodies with a combined improved anti-tumor activity is provided. The method comprising: (a) identifying binding epitopes of anti ErbB-2 antibodies; and (b) selecting a combination of at least two antibodies of the anti ErbB-2 antibodies exhibiting binding to different epitopes on the ErbB-2, at least one of the different epitopes being localized to a dimerization site of the ErbB-2, the combination of antibodies being with the combined improved anti-tumor activity. Also provided are novel antibody combinations uncovered according to the present teachings.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/063,940 filed on Mar. 15, 2011, which is a National Phase of PCTPatent Application No. PCT/IL2009/000816 having International FilingDate of Aug. 19, 2009, which claims the benefit of priority under 35 USC§119(e) of U.S. Provisional Patent Application No. 61/096,868 filed onSep. 15, 2008. The contents of the above applications are allincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA 072981 awardedby the NIH. The government has certain rights in the invention.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 60762SequenceListing.txt, created on Nov. 10,2014, comprising 1,577 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to antibodycombinations and use of same in treating cancer.

ErbB-2/HER2 is a member of the epidermal growth factor receptor (EGFR)family. When trans-activated, ErbB-2/HER2 stimulates several downstreamsignaling cascades, including the mitogen-activated protein kinasecascade [reviewed in (1)]. This ligand-less receptor is moderatelyexpressed in normal adult tissues, where it regulates cell growth anddifferentiation. By contrast, amplification of the corresponding geneand consequent overexpression of the HER2/ErbB-2 protein have beenreported in 20-30% of tumors of the breast (2-4) and ovary (4). Ingeneral, erbB-2 gene amplification associates with enhanced metastaticpotential and poor prognosis. Because ErbB-2 is expressed at relativelylow levels in normal tissues, it makes an attractive target forimmunotherapy. This was originally demonstrated in animals by Greene andcolleagues (5), who targeted Neu, the rodent form of ErbB-2, and laterdeveloped into a widely used clinical strategy [reviewed in (6)].Nevertheless, the molecular mechanisms underlying the growth inhibitoryeffects of anti-ErbB-2 monoclonal antibodies (mAbs) are not completelyunderstood. Several mechanisms of action have been proposed, includingimmune mechanisms, such as antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytototoxicity (CDC), increased cancercell apoptosis and interference with signaling cascades [reviewed in(6)].

Clinical studies established that Trastuzumab (Herceptin®), a humanizedmAb directed against ErbB-2, is active against ErbB-2-overexpressingmetastatic breast cancer, leading to its approval for clinical use (7).The objective response rates to Trastuzumab monotherapy is relativelylow (approximately 15%) and short lived (a median duration of 9 months)(8). On the other hand, mAbs seem to display a synergistic effect whencombined with chemotherapy, probably due to interruption ofErbB-2-driven survival pathways (9). Still another strategy, relevant topancreatic cancer, combines antibodies to EGFR and to ErbB-2 (10).

Yet another approach for improving the efficacy of antibody therapyrefers to the use of mAb combinations. Indeed a number of studies havebeen effected using at least two antibody combinations directed atdistinct epitopes of ErbB-2 [Drebin J. A. et al., Oncogene 2(3):273-277,1988; Kasprzyk et al., Cancer Res. 52(10):2771-2776, 1992; Harwerth etal., Br. J. Cancer 68(6):1140-1145, 1993; Spiridon et al. Clin. CancerRes. 8:1720-1730, 2002; Friedman et al. Proc. Natl. Acad. Sci. (2005)102:1915-1920; and U.S. patent application Ser. No. 11/342,615]

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture comprising a packagingmaterial identified for treating cancer packaging an anti-ErbB-2antibody comprising the CDR sequences of CNCM I-4112 (N12) and ananti-ErbB-2 antibody comprising the CDR sequences of L431 deposit numberCNCM I-4115.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture comprising a packagingmaterial identified for treating cancer packaging an anti-ErbB-2antibody comprising the CDR sequences of deposit number CNCM I-4112(N12) and an anti-ErbB-2 antibody comprising the CDR sequences of L26deposit number CNCM I-4113.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture comprising a packagingmaterial identified for treating cancer packaging an anti-ErbB-2antibody comprising the CDR sequences of N29 deposit number CNCM I-4114and an anti-ErbB-2 antibody comprising the CDR sequences of L26 depositnumber CNCM I-4113.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture comprising a packagingmaterial identified for treating cancer packaging an anti-ErbB-2antibody comprising the CDR sequences of N29 deposit number CNCM I-4114and an anti-ErbB-2 antibody comprising the CDR sequences of N12 depositCNCM I-4112.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture comprising a packagingmaterial identified for treating cancer packaging an anti-ErbB-2antibody comprising the CDR sequences of N29 deposit number CNCM I-4114and an anti-ErbB-2 antibody comprising the CDR sequences of L431 depositnumber CNCM I-4115.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture comprising a packagingmaterial identified for treating cancer packaging an anti-ErbB-2antibody comprising the CDR sequences of the antibody selected from thegroup consisting of N12 (CNCM I-4112), L431 (CNCM I-4115), N29 (CNCMI-4114) and L26 (CNCM I-4113) and an additional anti-ErbB-2 antibodydirected to a distinct epitope on ErbB-2, wherein when the anti-ErbB-2antibody is L26 the anti-ErbB-2 antibody directed to the distinctepitope on ErbB-2 is not Trastuzumab.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating cancer in a subject in needthereof, the method comprising administering to the subject an antibodycombination selected from the group consisting of: an anti-ErbB-2antibody comprising the CDR sequences of deposit number CNCM I-4112(N12) and an anti-ErbB-2 antibody comprising the CDR sequences of L431deposit number CNCM I-4115; an anti-ErbB-2 antibody comprising the CDRsequences of N12 deposit number CNCM I-4112 and an anti-ErbB-2 antibodycomprising the CDR sequences of L26 deposit number CNCM I-4113; ananti-ErbB-2 antibody comprising the CDR sequences of N29 deposit numberCNCM I-4114 and an anti-ErbB-2 antibody comprising the CDR sequences ofL26 deposit number CNCM I-4113; an anti-ErbB-2 antibody comprising theCDR sequences of N29 deposit number CNCM I-4114 and an anti-ErbB-2antibody comprising the CDR sequences of N12 deposit number CNCM I-4112;an anti-ErbB-2 antibody comprising the CDR sequences of N29 depositnumber CNCM I-4114 and an anti-ErbB-2 antibody comprising the CDRsequences of L431 deposit number CNCM I-4115; an anti-ErbB-2 antibodycomprising the CDR sequences of the antibody selected from the groupconsisting of N12 (CNCM I-4112), L431 (CNCM I-4115), N29 (CNCM I-4114)and L26 (CNCM I-4113) and an additional anti-ErbB-2 antibody directed toa distinct epitope on ErbB-2, wherein when the anti-ErbB-2 antibody isL26 the anti-ErbB-2 antibody directed to the distinct epitope on ErbB-2is not trastuzumab.

According to an aspect of some embodiments of the present inventionthere is provided a method of identifying a combination of antibodieswith a combined improved anti-tumor activity, the method comprising: (a)identifying binding epitopes of anti ErbB-2 antibodies; and (b)selecting a combination of at least two antibodies of the anti ErbB-2antibodies exhibiting binding to different epitopes on the ErbB-2, atleast one of the different epitopes being localized to a dimerizationsite of the ErbB-2, the combination of antibodies being with thecombined improved anti-tumor activity.

According to some embodiments of the invention, the packaging materialcomprises at least two containers for separately packaging theantibodies.

According to some embodiments of the invention, the article ofmanufacture further comprising a chemotherapy.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-F are graphs showing in vivo anti-tumor effects of antibodiesto ErbB-2/HER2 or combinations of same. Groups of seven CD-1/nude micewere injected subcutaneously with 5×10⁶ N87 cells. mAbs (total: 720 μgper animal) were then injected intraperitoneally, either alone or incombinations, at days 7, 10 and 13 post grafting. Saline-injected micewere used for control (open circles). Combination treatments using theindicated antibodies are shown (closed circles), along with mAb1 alone(closed triangles) and mAb2 alone (open triangles). Tumors were measuredonce a week using calipers, and the mean tumor volume (cm³±S.E.) wasplotted. Differences between the combined effects of N12+L431 or N12+L26versus the individual mAbs are statistically significant (p<0.05).

FIGS. 2A-G are graphs showing in vitro growth inhibitory effects ofantibodies to ErbB-2/HER2 and their combinations. Cultured N87 cellswere treated for 72 h with increasing concentrations of the indicatedantibodies (mAb1: closed triangles; mAb2: open triangles; HER indicatesHerceptin®/Trastuzumab) or their combinations (closed circles).Thereafter, the MTT assay was performed and cell viability presented aspercentage of control untreated cultures (±S.D.; n=8). Differencesbetween the combined effects of N12+L431 (>0.1 μg/ml), N12+L26 (>0.1μg/ml) and L26+Her (>0.6 μg/ml) versus the respective individual mAb arestatistically significant (p<0.05).

FIGS. 3A-D are graphs illustrating antibody displacement analyses. Theability of unlabeled mAbs to displace the indicated cell surface-bound¹²⁵I-mAb was used as a measure of the degree of antigenic overlap. N87cells were treated for 1 h at 4° C. with mAbs L431 (open circles), L26(closed triangles), N29 (open triangles) and N12 (closed circles). Theindicated radiolabeled mAbs (8 nM) were then added, and the cells wereincubated for additional 15 min before determination of cell-boundradioactivity. The experiment was repeated thrice.

FIGS. 4A-B are autoradiograms showing the effects of mAbs and theircombinations on the turnover rate of ErbB-2. N87 cells were incubatedfor 16 hrs with a mixture of [³⁵S]methionine and [³⁵S]cysteine and thenchased for the indicated periods of time with non-radioactive mediumcontaining 20 mg/ml (FIG. 4A) or the indicated lower concentrations(FIG. 4B) of anti-ErbB-2 mAbs. Residual ³⁵S-labeled ErbB-2 was subjectedto immunoprecipitation with a rabbit polyclonal antibody directed to thecarboxyl terminus of the protein and electrophoretically separated. Thenumbers below lanes represent densitometric quantification of ErbB-2relative to control, untreated cells.

FIGS. 5A-C are photographs showing the effects of antibody combinationson endocytosis and ubiquitinylation of ErbB-2, as evidenced by surfacebiotinylation. N87 cells were treated for either 8 h with the indicatedmAbs (5 mg/ml; A), or they were treated with the indicated antibodiesfor increasing time intervals (B). Thereafter, bound antibodies wereacid-stripped and the cell surface was biotinylated. Cells were thenlysed, ErbB-2 immunoprecipitates (IP) or total cell lysates (TCL)immunoblotted (IB) using the indicated antibodies, or streptavidinhorseradish peroxidase (HRP). Quantification of the signals is shownbelow the respective lanes. Ubiquitinylation of ErbB-2 (C) wasdetermined by immunoblotting (with an anti-ubiquitin antibody fromBabco/Covance) of ErbB-2 immunoprecipitates isolated from N87 cellstreated with different mAbs for 1 h at 37° C.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to antibodycombinations and use of same in treating cancer.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

While reducing the present invention to practice, the present inventorshave uncovered through laborious experimentation and screening that mAbsdirected at distinct epitopes of ErbB-2, especially epitopes involved indimerization, are highly effective anti-tumor agents in vivo when usedin combination. The superiority of these antibody combinations extendsto the ability to inhibit tumor cell growth in vitro, which weakensinvolvement of immune mechanisms. Conversely, effective antibodycombinations better than each antibody alone target surface ErbB-2 tointracellular degradation. These results suggest the use of antibodycombinations uncovered by the present teachings in cancer immunotherapyespecially for circumventing secondary drug resistance.

Thus, according to one aspect of the present invention there is provideda method of identifying a combination of antibodies with a combinedimproved anti-tumor activity.

The method according to this aspect of the present invention comprises:identifying binding epitopes of anti ErbB-2 antibodies; and selecting acombination of at least two antibodies of the anti ErbB-2 antibodiesexhibiting binding to different epitopes on the ErbB-2 polypeptide, atleast one of the different epitopes being localized to a dimerizationsite of the ErbB-2, the combination of antibodies being with thecombined improved anti-tumor activity.

As used herein “a combination of antibodies” refers to at least twodistinct antibodies, having different CDR sequences.

As used herein the phrase “anti tumor activity” refers to prevention oftumor formation and/or reduction of tumor size (e.g., volume) and/ormetastasis potential.

As used herein the phrase “combined improved anti tumor activity” refersto at least additive but preferably synergistically improved anti tumoractivity as explained hereinabove.

As used herein the term “synergy” refers a total affect that is greaterthan the sum of the individual contribution of each antibody.

As used herein “ErbB-2” refers to a receptor tyrosine kinase (RTK) ofthe epidermal growth factor receptor family, E.C. 2.7.10.1 also referredto as HER2, NEU and p185erbB-2.

Antibodies of this aspect of the present invention can be selected frompre-existing antibodies (e.g., publicly available hybridomas orrecombinant antibody libraries, further described hereinbelow) or fromnewly generated antibodies produced according to methods which arewell-known in the art and are further described hereinbelow.

Antibodies and methods of generating same are described at length in thefollowing sections.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)2, and Fv.These functional antibody fragments are defined as follows: (1) Fab, thefragment which contains a monovalent antigen-binding fragment of anantibody molecule, can be produced by digestion of whole antibody withthe enzyme papain to yield an intact light chain and a portion of oneheavy chain; (2) Fab′, the fragment of an antibody molecule that can beobtained by treating whole antibody with pepsin, followed by reduction,to yield an intact light chain and a portion of the heavy chain; twoFab′ fragments are obtained per antibody molecule; (3) (Fab′)2, thefragment of the antibody that can be obtained by treating whole antibodywith the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimerof two Fab′ fragments held together by two disulfide bonds; (4) Fv,defined as a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (5) Single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain and the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, incorporated herein by reference).

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)2. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al. [Proc.Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the VH and VLdomains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by [Whitlow andFilpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426(1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No.4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry[Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues form acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995).

As used herein, the term “epitope” refers to any antigenic determinanton an antigen to which the paratope of an antibody binds.

Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or carbohydrate side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics.

As mentioned the antibodies of the present invention bind differentepitopes on ErbB-2, of which one is located at an ErbB-2 dimerizationsite (i.e., binding of the antibody thereto is sufficient for blockingErbB-2 dimerization). The epitopes may be conformational or not and maybe overlapping or not, provided that the two antibodies cannot displaceeach other when they bind ErbB-2.

Methods of identifying the binding epitopes of antibodies are well knownin the art. Briefly, antibody binding epitopes can be determined by anantibody displacement assay. This may provide an initial understandingto the binding site. Antibody displacement techniques are well known inthe art and described in length in the Examples section which follows.Thus for instance, if the antibody tested is displaced by an antibodywhich is known to bind to ErbB-2 dimerization domain then it is mostlikely that the antibody in question binds to at least a partlyoverlapping epitope.

At times a finer analysis is required, to this end epitope mappingtechniques are employed. In this case the method of peptide scanning byReineke et al. 1999 is typically employed (Curr. Top. Microbiol.Immunol. 243:23-36). The entire antigen sequence [e.g., extracellularportion of ErbB-2 or dimerization domain i.e., Residues 195-320 ofErbB-2/HER2 (ARCKGP LPTDCCHEQC AAGCTGPKHS DCLACLHFNH SGICELHCPALVTYNTDTFE SMPNPEGRYT FGASCVTACP YNYLSTDVGS CTLVCPLHNQ EVTAEDGTQRCEKCSKPCAR VCYGLGMEHL); See Insights into ErbB signaling from thestructure of the ErbB2-pertuzumab complex. Franklin M C, et al, CancerCell 2004] is synthesized as linear 8 to 15 meric peptides that aresubsequently tested for binding the antibody in question. The peptidesmay be synthesized using methods which are well known in the art (e.g.,“SPOT” synthesis whereby the peptide is directly synthesized on a solidphase membrane e.g., cellulose). Solid-phase bound peptides are used forbinding studies directly on the membrane (heterogeneous assay). Twodifferent types of epitopes have to be considered: In linear(continuous) binding sites the key amino acids which mediate thecontacts to the antibody are located within one part of the primarystructure, usually not exceeding 15 amino acids in length. Peptidescovering these sequences have affinities to the antibody which arewithin the range shown by the entire protein antigen. In discontinuous(conformational) binding sites the key residues are distributed over twoor more binding regions which are separated in the primary structure.Upon folding, these binding regions are brought together on the proteinsurface to form a composite epitope. Substitutional analysis istypically effected to characterize epitopes which comprise amino acidswhich are buried in the antigen structure or when a side chainmodification (e.g., glycosylation) is part of the epitope structure.

Once the binding properties of the antibodies are characterized, acombination of at least two antibodies of the anti ErbB-2 antibodiesexhibiting binding to different epitopes on the ErbB-2 (as describedhereinabove), is selected.

Those antibody combinations selected according to the present teachingscan be further qualified having anti tumor activity using in vitro andin vivo methods which are well known in the art and described at lengthin the Examples section which follows.

While implementing the present teachings, the present inventors havesuccessfully uncovered combinations of antibodies which are mosteffective in treating cancer in a subject in need thereof.

N12, L26, N29 and L431 have been deposited in the Collection Nationalede Cultures de Microorganismes INSTITUT PASTEUR 25, Rue du Docteur RouxF-75724 Paris CEDEX 15. Antibodies have been deposited under theBudapest Treaty. The materials have been registered on Jan. 13, 2009.

The registration numbers are as follows:

N12 CNCM I-4112;

L26 CNCM I-4113;

N29 CNCM I-4114;

L431 CNCM I-4115.

Thus the present invention, in some embodiments thereof, provides for anarticle of manufacture which comprises a packaging material identified,in print, for treating cancer. The packaging material comprises anycombination of the following antibodies:

an anti-ErbB-2 antibody comprising the CDR sequences of N12 depositnumber CNCM I-4112 and an anti-ErbB-2 antibody comprising the CDRsequences of L431 deposit number CNCM I-4115;an anti-ErbB-2 antibody comprising the CDR sequences of N12 depositnumber CNCM I-4112 and an anti-ErbB-2 antibody comprising the CDRsequences of L26 deposit number CNCM I-4113;an anti-ErbB-2 antibody comprising the CDR sequences of N29 depositnumber CNCM I-4114 and an anti-ErbB-2 antibody comprising the CDRsequences of L26 deposit number CNCM I-4113;an anti-ErbB-2 antibody comprising the CDR sequences of N29 depositnumber CNCM I-4114 and an anti-ErbB-2 antibody comprising the CDRsequences of N12 deposit number CNCM I-4112;an anti-ErbB-2 antibody comprising the CDR sequences of N29 depositnumber CNCM I-4114 and an anti-ErbB-2 antibody comprising the CDRsequences of L431 deposit number CNCM I-4115;an anti-ErbB-2 antibody comprising the CDR sequences of the antibodyselected from the group consisting of N12 (CNCM I-4112), L431 (CNCMI-4115), N29 (CNCM I-4114) and L26 (CNCM I-4113) and an additionalanti-ErbB-2 antibody directed to a distinct epitope on ErbB-2, whereinwhen said anti-ErbB-2 antibody is L26 said anti-ErbB-2 antibody directedto a distinct epitope on ErbB-2 is not trastuzumab.

As used herein the term “subject” refers to a mammal, preferably a humansubject.

As used herein the term “treating” refers to alleviating or diminishinga symptom associated with a disease (e.g., cancerous disease).Preferably, treating means cures, e.g., substantially eliminates, thesymptoms associated with the disease.

As used herein the term “cancer” refers to a tumoral disease whichdepends on ErbB-2 (activity and/or expression) for onset and/orprogression. Examples of cancer which can be treated in accordance withthe present teachings include, but are not limited to, carcinoma,adenocarcinoma, lung cancer, liver cancer, colorectal cancer, brain,head and neck cancer (e.g., neuro/glioblastoma), breast cancer, ovariancancer, transitional cell carcinoma of the bladder, prostate cancer,oral squamous cell carcinoma, bone sarcoma, biliary tract cancer such asgallbladder carcinoma (GBC), kidney cancer and pancreatic cancer.

Antibodies of the present invention can be administered to an organismper se, or in a pharmaceutical composition where they are mixed withsuitable carriers or excipients (either individually or in aco-formulation).

As used herein, a “pharmaceutical composition” refers to a preparationof one or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

As used herein, the term “active ingredient” refers to the antibodiesaccountable for the intended biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier,” which may be usedinterchangeably, refer to a carrier or a diluent that does not causesignificant irritation to an organism and does not abrogate thebiological activity and properties of the administered compound. Anadjuvant is included under these phrases.

Herein, the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils, and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found inthe latest edition of “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., which is herein fully incorporated byreference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal, or parenteraldelivery, including intramuscular, subcutaneous, and intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations that can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries as desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, and sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents, such ascross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane, or carbon dioxide. In the case of apressurized aerosol, the dosage may be determined by providing a valveto deliver a metered amount. Capsules and cartridges of, for example,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base, such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with, optionally, anadded preservative. The compositions may be suspensions, solutions, oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing, and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water-based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters such as ethyl oleate, triglycerides, orliposomes. Aqueous injection suspensions may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe active ingredients, to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., a sterile, pyrogen-free,water-based solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, for example, conventional suppository bases such as cocoabutter or other glycerides.

Pharmaceutical compositions suitable for use in the context of thepresent invention include compositions wherein the active ingredientsare contained in an amount effective to achieve the intended purpose.More specifically, a “therapeutically effective amount” means an amountof active ingredients (e.g., a nucleic acid construct) effective toprevent, alleviate, or ameliorate symptoms of a disorder (e.g.,ischemia) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, the dosage orthe therapeutically effective amount can be estimated initially from invitro and cell culture assays. For example, a dose can be formulated inanimal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration, and dosage canbe chosen by the individual physician in view of the patient'scondition. (See, e.g., Fingl, E. et al. (1975), “The PharmacologicalBasis of Therapeutics,” Ch. 1, p. 1.)

Dosage amount and administration intervals may be adjusted individuallyto provide sufficient plasma or brain levels of the active ingredient toinduce or suppress the biological effect (i.e., minimally effectiveconcentration, MEC). The MEC will vary for each preparation, but can beestimated from in vitro data. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks, oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Typically used models for analyzing the effect of the agents describedherein on tumors are provided infra.

An animal model for ErbB-2 induced gallbladder carcinoma is described inKawamoto et al. Cellular, Molecular, and Tumor Biology 89: Mouse Modelsof Prostate and Gastrointestinal Cancers Abstract Number 4313.

A mouse model for ErbB-2 dependent breast cancer is described in Liu etal. Cancer Research 65, 5325-5336, 2005.

Agus et al., “Response of Prostate Cancer to Anti-Her-2/neu Antibody inAndrogen-dependent and -independent Human Xenograft Models” CancerResearch 59:4761-4764 (1999).

Kasprzyk et al., “Therapy of an Animal Model of Human Gastric CancerUsing a Combination of Anti-erbB-2 Monoclonal Antibodies” CancerResearch 52(10):2771-2776 (May 15, 1992).

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA-approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser device may also be accompaniedby a notice in a form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may includelabeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a preparation of the invention formulated in apharmaceutically acceptable carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition, as further detailed above.

It will be appreciated that the antibodies of the present invention canbe provided to the individual with additional active agents to achievean improved therapeutic effect as compared to treatment with theantibodies alone. In such therapy, measures (e.g., dosing and selectionof the complementary agent) are taken to adverse side effects which maybe associated with combination therapies.

Administration of such combination therapy can be simultaneous, such asin a single capsule having a fixed ration of these active agents, or inmultiple capsules for each agent.

Thus, for example, the antibodies of the present invention can beadministered along with analgesics, chemotherapeutic agents (e.g.,anthracyclins), radiotherapeutic agents, hormonal therapy and othertreatment regimens which are well known in the art.

It is expected that during the life of a patent maturing from thisapplication many relevant antibodies will be developed and the scope ofthe term of the patent is intended to include all such new technologiesa priori.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”. This termencompasses the terms “consisting of” and “consisting essentially of”.

The phrase “consisting essentially of” means that the composition ormethod may include additional ingredients and/or steps, but only if theadditional ingredients and/or steps do not materially alter the basicand novel characteristics of the claimed composition or method.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate some embodiments of the invention in anon limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Materials and Experimental Procedures

Materials, Antibodies and Cells.

Unless indicated, materials were purchased from Sigma (St. Louis, Mo.),cells from the American Type Culture Collection and antibodies fromSanta Cruz Biotechnology (Santa Cruz, Calif.). Radiochemicals werepurchased from Amersham (Buckinghamshire, UK). Trastuzumab was providedby Genentech (South San Francisco, Calif.). Generation of mAbs to ErbB-2(i.e., N29, L431, N12 and L26) was previously described in Klapper L N,Vaisman N, Hurwitz E, Pinkas Kramarski R, Yarden Y, Sela M (1997) Asubclass of tumor-inhibitory monoclonal antibodies to ErbB-2/HER2 blockscrosstalk with growth factor receptors. Oncogene 14: 2099-2109; andStancovski I, Hurwitz E, Leitner O, Ullrich A, Yarden Y, Sela M (1991)Mechanistic aspects of the opposing effects of monoclonal antibodies tothe ERBB2 receptor on tumor growth. Proc Natl Acad Sci USA 88:8691-8695. The antibodies were purified on protein G Plus-Aagarose.

Surface Biotinylation Assay.

Cells were incubated at 37° C. with mAbs and then transferred to ice andmAbs removed using a low pH solution (0.15 M acetic acid containing 0.15M NaCl; 4 min). The cells were washed and incubated for 60 min at 4° C.with N-hydroxysuccinimide (NHS)-biotin (0.5 mg/ml; Calbiochem). Couplingof biotin was blocked with 15 mM glycine (15). Thereafter, cells weresolubilized by the addition of lysis buffer [50 mM HEPES (pH 7.5), 150mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mMphenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 0.5 mM Na₃VO₄, 5μg/ml leupeptin and 5 μg/ml aprotonin] and a rabbit antibody to ErbB-2was used for immunoprecipitation.

Radiolabeling of Antibodies.

MAbs (100 μg in 0.2 ml PBS) were radiolabeled using Na¹²⁵I [5 μl; 0.5mCi (18.5 MBq)] and chloramine-T (10 mg/10 μl). The reaction mixture waschromatographed on Sephadex G-25 yielding radiolabeled mAbs of 1-2mCi/mg protein. MAb N29 was radiolabeled by using the[¹²⁵I]-Bolton-Hunter reagent (PerkinElmer Sciences, Inc; Boston, Mass.).

Antibody Competition Assay.

N87 cells (250,000 cells/well) grown in 24-well plates were treated for1 h at 4° C. with increasing concentrations of unlabeled mAbs.Radiolabelled mAbs (8 nM) were then added, and the cells incubated foradditional 15 min at 4° C. After washing, the cells were solubilized in0.5 N NaOH solution, prior to determination of radioactivity.

Inhibition of N87 Tumor Cell Growth in Culture.

Antibodies were added to N87 cells (10,000 cells/well) grown in 96-wellplates. Incubation at 37° C. proceeded for 72 h and cell viabilitydetermined by using the MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reagent(18).

Determination of the Effect of Antibodies on Receptor Turnover.

N87 cells were labeled by incubation (16 h at 37° C.) in methionine- andcysteine-free medium supplemented with 10% dialyzed fetal calf serum andPromix, a mixture of [³⁵S]methionine and [³⁵S]cysteine (50 μCi/ml).Thereafter, cells were chased for various periods of time by incubationin fresh medium in the absence or presence of the antibodies. The cellswere then washed, and lysates subjected to immunoprecipitation.

Tumorigenic Cell Growth in Animals.

Female CD/nude mice were subcutaneously injected with N87 cells(5×10⁶/mouse). mAbs were injected intraperitoneally at days 7, 10 and 13post grafting. Groups of 7 mice were used, and each mouse received 0.72mg of purified mAb. Tumor parameters were measured once a week.

Statistical Analysis.

Student's t-test (two-tailed) was used to test differences between theeffects of antibody combinations and single treatment. Values of p<0.05were considered statistically significant.

Example 1 Combinations of Monoclonal Anti-ErbB-2 AntibodiesCollaboratively Inhibit Tumor Growth In Vivo

To examine the effect of combining two antibodies, N87 cells weresubcutaneously injected into athymic mice, which elicited rapidlygrowing tumors. Thereafter, the four mAbs or their six combinations wereintraperitoneally injected into groups of seven mice. FIGS. 1 a-f depictthe average tumor volume of each group as a function of post-graftingtime. While the antibodies differed in their therapeutic efficacy, withonly one exception (a combination of mAbs L431 and L26, FIG. 1 d),antibody combinations more effectively inhibited tumor growth than eachantibody alone. Notably, tumors were completely eradicated in at leastfour of seven mice after treatment with the two most effectivecombinations, namely: L26 plus N12 and L431 plus N12. Moreover, thiseffect persisted six weeks after the last injection. Interestingly, whensingly applied, N29 was not effective. Nevertheless, this mAb enhancedthe inhibitory effect of other mAbs, but tumors initially inhibited byN29-containing combinations displayed re-growth (FIGS. 1 a-b, f). Inconclusion, the majority of antibody combinations tested showed clearsynergistic anti-tumor effects.

Example 2 Antibody Combinations Inhibit Cell Growth In Vitro

The synergistic in vivo effects observed may be due to immunologicalmechanisms (e.g., ADCC). Hence, it is expected that antibody synergywould not extend to an in vitro test, such as the MTT cell proliferationassay. FIGS. 2 a-g depict the results of an assay performed with N87cells incubated for 72 h with various antibodies and their combinations.Remarkably, strong correlation was found with most antibody combinationsbetween the results obtained in this assay and the in vivo experiments(FIGS. 1 a-f): four of the six combinations exhibited synergisticeffects. Moreover, the relative potency of the various combinations waspreserved in vitro. For instance, the superior antibody combination inanimals, namely N12+L431, was also the most potent in vitro combination,yielding 54% reduction in cell proliferation already at 0.3 μg/ml.Likewise, our least potent in vivo combination, namely L26+L431,displayed no synergy in vitro. Notably, the N29 antibody elicited noinhibitory effects both in vitro and in animals, and when tested incombinations it did not improve the effects of mAbs N12 and L431. Yet,this antibody reproducibly enhanced the in vitro anti-proliferativeeffect of L26 (FIG. 2 f), which attributes a role for antigencross-linking by a second antibody.

We previously reported that a combination of L26, an antibody capable ofinhibiting heterodimerization of ErbB-2 (16), and Trastuzumab displayssynergy in an in vivo anti-tumor test (15). Extension of this analysisis shown in FIG. 2 g: the combination is significantly more potent thaneach antibody alone. To exclude complement involvement, the MTT assaywas repeated using a serum replacement mixture containing heat-treatedalbumin Because the synergistic effects of antibody combinations wereobserved also in the absence of serum (data not shown), it is concludedthat neither CDC nor ADCC contribute to the ability of anti-ErbB-2antibodies to collaborate in vitro. Taken together, the overallsimilarity between the in vivo and in vitro effects of antibodycombinations implies that the synergistic anti-growth effects aremediated by activities intrinsic to the antibody molecules.

Example 3 Characterization of Epitope Sharing by Anti-ErbB-2 Antibodies

Previous analysis of a panel of antibodies to EGFR indicated that mAbsynergy requires interactions with two mutually distinct antigenicdeterminants (15). Therefore, the ability of each anti-ErbB-2 mAb todisplace radiolabeled versions of the other mAbs, was analyzed. Theresults shown in FIG. 3 confirmed that the non-synergizing pair ofantibodies, namely L26 and L431, is cross-competitive. On the otherhand, pairs of mAbs that displayed synergy both in vivo and in vitro,including L431 plus N12 and L26 plus N12, bind distinct epitopes ofErbB-2. In the case of N29, the unlabeled antibody did not interferewith the binding of any of the radiolabeled antibodies, while thebinding of a radiolabeled derivative of N29 was reduced by L431,possibly through steric interference. Consistent with this possibility,an attempt to map the N29 epitope has failed, suggesting that N29recognizes a carbohydrate-containing epitope (19).

Example 4 Specific Antibody Combinations Accelerate Removal of ErbB-2from the Cell Surface

The interaction of receptor tyrosine kinases with their respectiveligands is often coupled to rapid endocytosis and receptor degradationin lysosomes. It was shown that mAbs can induce an analogous, albeitslower effect (20), which is associated with inhibition of tumorigenesis(12). Therefore, the potential of the present sets of mAbs to alter theturnover rate of ErbB-2 was tested. To this end, N87 cells werebiosynthetically labeled and then chased in fresh, mAb-containingmedium. As shown in FIG. 4A, among the four mAbs tested, L431 remarkablyaccelerated degradation of ErbB-2, and this was slightly enhanced by theaddition of N12. Because the non-synergistic L26+L431 combination wasless effective, various concentrations of each antibody were tested, aswell as the two combinations (FIG. 4B; chase of 2 h). The resultsconfirmed that the L431+N12 combination is superior at a relatively lowantibody concentration, but this difference became less apparent athigher concentrations.

Because pulse-chase analysis examines the overall pool of ErbB-2, butmAbs interact with only the surface-exposed receptors, surfacebiotinylation was applied. Cells were surface-labeled with biotinfollowing incubation for 8 hours with single mAbs, or theircombinations. Thereafter, antibodies were acid-stripped and ErbB-2immunoprecipitated. As depicted in FIG. 5A, the combination of mAbs L431and N12 most potently down-regulated ErbB-2 from the cell surface.Interestingly, the other combination, L431+L26, was less effective thanL431 alone, in line with their competitive interactions (FIGS. 3 a-d).The superiority of the L431+N12 combination is evident also from thetime course of ErbB-2 down-regulation (FIG. 5B): almost all surfaceErbB-2 molecules were cleared from the cell surface following a 24-hlong incubation with the L431+N12 combination, but each antibody exertedonly a limited effect.

Sorting of receptor tyrosine kinases (e.g., EGFR) for intracellulardegradation involves their ubiquitinylation, which recruits to theinternalizing receptor ubiquitin-binding coat adaptors, such as Epsin[reviewed in (21)]. It was proposed that this mechanism may underliemAb-induced degradation of ErbB-2 (22). Hence, by employinganti-ubiquitin antibodies the ability of mAbs and their combinations toenhance ErbB-2 ubiquitinylation was addressed. This assay revealed weak,but reproducible mAb-induced ubiquitinylation of ErbB-2. Interestingly,L431 displayed higher effects than other mAbs, especially when combinedwith the non-competitive N12 mAb. Once again, when combined with thecompetitive L26 antibody, the ubiquitinylation effect of L431 decreased.In conclusion, certain mAbs enhance ubiquitinylation of ErbB-2 andeffectively target the surface-localized sub-population of this receptorto intracellular degradation. Combining two non-competitive mAbsenhances this activity, which correlates with the synergistic growthinhibitory effects of such combinations (FIGS. 1 a-f and 2 a-g)

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

REFERENCES Other References are Cited Throughout the Application

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What is claimed is:
 1. A method of treating pancreatic cancer in asubject in need thereof, the method comprising administering to thesubject an anti-ErbB-2 antibody comprising the CDR sequences of N12deposit number CNCM I-4112 and an anti-ErbB-2 antibody comprising theCDR sequences of L26 deposit number CNCM I-4113; wherein the combinationof antibodies being with a combined improved anti-tumor activity.
 2. Themethod of claim 1, wherein said administering comprises intravenousadministration.
 3. The method of claim 1, wherein said anti-ErbB-2antibodies are in a co-formulation.
 4. The method of claim 1, whereinsaid anti-ErbB-2 antibodies are in separate formulations.
 5. The methodof claim 1, wherein said subject is a human subject.